Acta Protozool. (2015) 54: 233–240 www.ejournals.eu/Acta-Protozoologica Acta doi:10.4467/16890027AP.15.019.3216 Protozoologica

Apolocystis proventus sp. nov. (Apicomplexa: Monocystinae) a New Species of Aseptate Gregarine from Egyptian Earthworms: Pheretima californica and Pheretima elongata (Annelida: Oligochaeta)

Nadia RAMADAN, Samia FAWZY, Medhat ALI, Ahmed NIGM

Department of Zoology, Faculty of Science, Ain Shams University, Abbassia, Cairo, Egypt

Abstract. A new acephaline gregarine is described from the earthworms Pheretima californica and Pheretima elongata. The gregarine was either embedded in the pharyngeal glandular tissue or found free in the coelomic fluid around the pharyngeal region in front of the crop. Adult trophozoites measured 48–65 µm in diameter and are mostly active with a wavy pellicle. Heterogeneity in the endoplasm of active trophozoites was observed. Gametocysts measured 56–81 µm in diameter, with a characteristic thick cyst wall. Navicular sporocysts mea- sured 5.8 ± 0.2 × 3.5 ± 0.4 µm, with small truncate plugs.

Key words: Gregarine, Pheretima, Coelom, Apolocystis, Pharyngeal glands, Apicomplexa and Monocystinae.

INTRODUCTION cystis by Cognett de Martiis (1923). Several species of Apolocystis were described (Bahatia and Setna 1926, Phillips and Mackinnon 1946, Ramadan 1969, Levine Monocystids belonging to the eugregarines are par- 1977, Segun 1978, Pradhan and Dasgupta 1983, Ar- asitic protozoa frequently infecting the seminal vesi- mendáriz and Gullo 2002, Bandyopadhyay et al. 2004, cles and coelomic cavities of many oligochaetes. Most Bhowmik et al. 2012). In this paper, a new species of published papers, which described those parasites, are Apolocystis is described with comparative comments taxonomic in nature or check lists of monocystid spe- on closely related species. cies along with their related hosts (Ruston 1959, Marek 1967, Segun 1971a, b, Levine 1977 and 1988, Pizl 1989a, b and Frolov 1991). All species belonging to MATERIALS AND METHODS genus Monocystis, characterized by round trophozoites with no polarity, were included into the genus Apolo- The host worms Earthworms were collected from Maghagha region (Menia Address for correspondence: Medhat Ali, Department of Zoology, Governorate) and the Zoological Gardens (Giza Governorate), Faculty of Science, Ain Shams University, Abbassia PO box 11566, Egypt. Specimens collected were stored into soil-filled plastic con- Cairo, Egypt; E-mail: [email protected]; Fax: 26842123 tainers and transferred live to the laboratory of invertebrates, depart- 234 N. Ramadan et al. ment of zoology, faculty of science, Ain shams university. Some Genus: Apolocystis (Cognetti de martiis 1923) earthworms were dissected, and the seminal vesicles were carefully Description: sub-spherical, oval, parasite is embed- removed and placed on a clean grease-free slide with a drop of 0.8% ded into the pharyngeal glandular tissue or free in the NaCl solution. A thin film of seminal fluid was pipette onto the slides and saved with glass cover slips. Wet mounts were examined coelomic fluid around the pharyngeal region in front of for living protozoans under a compound microscope. The coelomic the crop region. Adult trophozoites have no polar dif- fluid of the examined worms was withdrawn by a micropipette and ferentiation (48–65 µm in diameter). Most trophozoites then placed on clean slides with a drop of 0.8% NaCl solution and are active with wavy pellicle. Heterogeneity (irregular covered with glass cover slips for examination. After initial exam- distribution of paraglycogen granules) in the endoplasm ination of living protozoans, slides were semidried and fixed for of active trophozoites is observed. Gametocysts meas- 20 minutes in Schaudin’s fluid (saturated aqueous solution of mer- curic chloride and glacial acetic acid). Slides were then treated in ure 56–81 µm in diameter, with a characteristic thick ethyl alcohol for removal of excess of mercuric chloride. cyst wall. Navicular sporocysts measure 5.8 µm ± 0.2 × 3.5 µm ± 0.4, with small truncate plugs. Histological preparations Remarks: The parasite was found embedded in the For histological examination, sectioned tissues were prepared pharyngeal glands, (Fig. 1) free in the coelom, and in by fixing body segments of infected worms in Bouin’s fluid for front of the crop region of Pheretima californica and 24 hours. Specimens were then transferred to a mixture of 70% ethyl P. elongata. Growing trophozoites were mainly round alcohol and lithium carbonate, then dehydrated through an ascend- ing series of alcohols and cleared in terpineol for 3 days. Specimens or oval and had no apparent polarity. The pellicle exhib- were embedded in parablast wax and sectioned (5 µm thickness) on ited active short contractility waves which took many clean slides. Slides were then dewaxed in xylene, then rehydrated forms (Fig. 2). The endoplasm of the growing tropho- through a descending series of alcohols to distilled water. A subset zoites exhibited more heterogeneity than adults. Many of slides were transferred to Mallory I solution (1% Acid fuchsin) growing trophozoites had endoplasmic areas without for 15 seconds, rinsed in distilled water to differentiate red color of paraglycogen granules. In fresh preparations, the en- fuchsin for 10 seconds, treated with Phosphomolybdic acid (1%) for 1–5 minutes, then stained with Mallory II solution (equal parts of doplasm of some adult trophozoites exhibited an ir- 1% Aniline blue and 4% Orange G) for 2 minutes. Differentiation regular darker area (more aggregation of paraglycogen was done with 90% ethyl. Slides were then washed in distilled wa- granules) around the nucleus with narrow radiating ter, dehydrated in an ascending series of alcohols, cleared in xylene, extensions (Fig. 3). Paraglycogen granules were oval and mounted in Canada balsam. The other subset of slides were to elongate in shape with rounded ends and ranged stained in haematoxylin for 30 minutes, transferred to a tap water (1 minute), and counter stained in eosin. Slides were dehydrated in from 0.3 µm to 1.6 µm in length. The nucleus was de- ethyl alcohol, cleared and mounted. Photomicrographs were taken formed (irregular in shape) in most trophozoites due to by a Kodak digital camera (model 1450Z) attached to a compound the pressure exerted by the active internal streaming of microscope. the endoplasm. The nucleus was round, eccentric, and measured about 8–12 µm in diameter (Fig. 4). Karyo- somes were relatively large, eccentric and about 2.4– RESULTS 5.7 µm in diameter (Fig. 4). The adult trophozoites had no polar differentiation and measured about 48–65 µm in diameter (Table 1). Apolocystis proventus sp. nov. Gametocysts had sizes ranged from 56 to 81 μm in Phylum: Apicomplexa (Levine 1977) diameter (Fig. 5). The cyst wall is characterized by its Order: Eugregarinida (Leger 1900) considerable thickness. However, the morphological Family: Monocystidae (Butschli 1882) change of the parasite inside the cyst was hard to ob- Subfamily: Monocystinae Grassè, 1953 (= Mono- serve during progressive stages. In sectioned material, cystidae Bhatia, 1930) the cyst wall was deeply stained with Haematoxylin

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Figs 1–6. Apolocystis proventus a new greagarine from Egyptian earthworms. 1 – Section of the host worm showing a parasite in gametocyst stage (arrowhead) embedded in pharyngeal glands (PG), Mallory stain. Note-Insert showing a magnified gametocyst.2 – An active growing trophozoite showing the waves of contractility (arrowheads), fresh preparation. 3 – A growing trophozoite showing an irregular darker area (more aggregation of granules) around the nucleus (N) with narrow radiating extensions (white arrowhead), fresh preparation. 4 – Adult trophozoite showing the nucleus (N) and karyosome (K), fresh preparation. 5 – A mature gametocyst filled with sporocysts (S), fresh prepa- ration. 6 – A section of immature gametocyst showing a thick gametocyst wall (G W), Haematoxylin and eosin stains. A New Aseptate Gregarine from Egyptian Earthworms 235 236 N. Ramadan et al.

Table 1. Average size (µm) of the observed Apolocystis proventus stages in coelom of Pheretima californica and P. elongata.

Stage Average (µm) Range (µm) Standard deviation (± µm) Trophozoite – Cell 57.7 48–65 6.3 – Nucleus 10.3 8–12 1.7 – Karyosome 4 2.4–5.7 1.6 – Granule 0.9 0.3–1.6 0.6

Gametocyst – Diameter 73 56–81 5.9

Sporocyst – Length 5.8 5.6–6 0.2 – Width 3.5 3.1–3.9 0.4

and eosin (Fig. 6). Sporocysts are navicular, with small Fig. 7. A single sporocyst, note the two small flat plugs (P), fresh truncate plugs at both ends, and measured 5.8 ± 0.2 µm preparation. in length and 3.5 ± 0.4 µm in width (Fig. 7).

DISCUSSION In the present parasite, the endoplasm of the grow- ing trophozoite exhibited more heterogeneity than that of the adult trophozoite. The growing trophozoites had The parasite described herein belongs to the genus areas of their endoplasm devoid of paraglycogen gran- Apolocystis. The spheroidal form and lack of polarity in ules; with progressive growth of the trophozoite and its trophozoites are evident characteristics. One species, more production of paraglycogen granules, the degree A. aggregata was described and named by Ramadan of heterogeneity in the endoplasm decreased. General- (1969), from two Egyptian earthworms Pheretima cali- ly, trophozoites related to Ramadan’s work were much fornica and Pheretima hawayana (Table 2). Apart from larger in its measurements (about double) than those of the difference in trophozoite size, and larger nuclei of the present gregarine. (Table 2) A. aggregata, and the gregarine describe herein, a spe- Within the range size of trophozoites of the present cial character was exhibited by A. aggregata. Young parasite, were those of A. beaufortii (Cognetti de Mar- trophozoites displayed the tendency to aggregate in tiis 1918) (50 μm); A. chotonagpurensis Bandyopad- groups consisting of 5 to 20 individuals. Moreover, the hyaye et al., 2004 (42 to 83 μm); A. minima (Boisson size and shape of the sporocysts in A. aggregata dif- 1957) Frolov 1991 (60 μm); and A. minuta Troisi, fered from these of the present species. Apolocystis ag- 1933 (40 to 46 μm). A. beaufortii was collected from gregata sporocysts are navicular with pointed ends and the seminal vesicles of Pheretima beaufortii. It was measured 10 µm × 3.5 µm, while in the present species characterized by the formation of two morphologically the sporocysts are navicular with small truncated ends distinguishable types of gametes (anisogamy). Apolo- and measured 5.8 µm (±0.6 µm) × 3.5 µm (±0.4 µm). cystis chotonagpurensis was collected from the seminal An Apolocystis sp. was reported by Ramadan (1969) vesicles of Amynthasrobusta (earthworm). It was char- in an Egyptian oligochaete species; only trophozoites acterized by very fine rows of cytoplasmic processes were observed. This parasite was exhibiting a striking and large nucleus if compared with the diameter of feature; as the endoplasm of trophozoite takes the form our present trophozoite. Apolocystis minima was col- of small vacuoles surrounded by moderately coarse lected from the seminal vesicles of P. posthuma. It was granules, while the vacuoles themselves are occupied characterized by anisogamy, a central karyosome, and by much finer granules. This description may be com- larger paraglycogen granules (up to 3 μm) compared to parable with some previously described aspects related those of the present parasite (0.3 to 1.6 μm); it also has to the endoplasm of the present parasite. much longer sporocysts (twice the length of those in the A New Aseptate Gregarine from Egyptian Earthworms 237

Table 2. Comparison among three Apolocystis species infecting some earthworms in Egypt.

Parasite Host and Localities Measurements Reference Trophozoite Nucleus Karyosome Gametocysts Sporocysts Apolocystis Pheretima californica 60 µm–100 µm 13 µm–22.5 µm 3 µm–5 µm 130 µm–180 µm Navicular, Ramadan aggregata and (76.5 µm) Mainly eccentric (175 µm) pointed ends 1969 Ph. hawayana Young troph. 10 µm × 3.5 µm Zoological and in groups Orman botanical Garden

Apolocystis Unidentified species 90 µm–115 µm 32 µm–35 µm 7 µm – – Ramadan sp. of oligichaetes Mostly eccentric 1969 Abou-Rawash

Apolocystis Pheretima californica Round: 8 µm–12 µm 2.4 µm–5.7 µm Round: Navicular, very Present proventus and 48 µm–65 µm Mainly eccentric Eccentric 56 µm–81 µm small truncated work n. sp. Ph. elongata 57.7 µm (±6.3 µm) 73 µm (±5.9) ends Zoological Garden 5.8 µm (±0.2) × and Maghaga region 3.5 µm (±0.4) (Menia Governorate)

present study; 10.5 and 5.8 μm, respectively). Apolo- (1909) described as many thin membranes formed by cystis minuta was collected from the seminal vesicles the host worm itself. Moreover, some trophozoites of Lumbricus terrestris, L. castaneus and L. rubellus. It could be occasionally found free and might be attached was characterized by being the smallest species belong- to the nephredia. This species was characterized by ing to genus Apolocystis, with 3 sizes of sporocysts and the syzygy stage which involved 2 to 3 gamonts, up to had very large paraglycogen granules (up to 5 to 7 μm) 16 sporocysts in the gametocysts, and the sporocysts (Table 3). with swollen equatorial planes (Table 3). Apart from the difference in morphometric param- Young trophozoites of A. janovyi are intercellular par- eters of trophozoites, there were other monocystid gre- asites of the oesophageal epithelium, while all remain- garines belonging to genus Apolocystis which were ing stages of the life cycle are attached to the parietal recorded in the coelom of various species of oligocha- epithelium and the visceral peritoneum of the post-cli- etes and exhibited more or less behavioral similarities tellar segments up to the posterior end of the host. Gen- with the present parasite. It was recorded that in four erally, measurements of different stages of A. janovyi, species of Apolocystis, some stages of their life cycle were triple in size compared to those of the correspond- were involved into certain tissues of their host worms; ing stages of A. proventus. Many authors described and A. laverensis Rees, 1963, A. michaelseni Hesse, 1909, reported A. herculea (Bosanquet 1894, Hesse 1909, A. janovy Armendariz and Gullo, 2002 and A. herculea Ress 1963, Segun 1978 and Pizl 1990). Bosanquet Bosanquet, 1894. (1894) reported that the infection was restricted to the Young trophozoites of A. lavernensis develope in posterior region of the host worm. Young trophozo- the ovisac and exhibited high activity in their move- ites and gametocysts were embedded in the splanchnic ments. Growing and mature trophozoites, (the syzygy epithelial layer. The growing and mature trophozoites stages and gametocysts) were free in the coleomic cavi- were mainly free in the coelomic cavity, or to a less ties of the genital segments. And mature trophozoites extent, attached to nephredia, the coelomic epithelium had no observable activity in their movement. All life of the intestine and of the body wall by what Bosan- cycle stages of A. michaelseni were attached to the peri- quet described as double membranes. Hesse (1909) toneal epithelium of the body wall and the splanchnic agreed with what was described by Bosanquet (1894) epithelial cells belonging to post-clitellar segments, This except that the gametocysts were phagocytized by host attachment was attributed by the help of what Hesse amoebocytes and found in groups. Segun (1978) found 238 N. Ramadan et al. 1923 Rees 1963 Troisi 1933 Troisi Hesse 1909 Segun 1971 Biswas 2004 (Frolov 1991) Bandyopadhyay, Bandyopadhyay, The present work Roychoudhuri and Loubatieres (1955) Cognetti de Martiis – – × × Cyst 68–74 71–96 Round: 100 um In coelom 250 × 56–81 µm 55–65 µm 46–83 µm 235–300 × anisogamy Boisson 1957 anisogamy 330–540 μm 73 µm (±5.9) 170–220 µm, as maximum. with 16 spores – × × ends 6.5–7 27–31 11 × 5.5 11 10.5 long 3.5–4 µm Sporocyst Gametocyst Reference flat button 12–16 µm oval spore, round ends 12 µm long With flat ends With with remarkable equatorial plane. (±0.4) Navicular, (±0.4) Navicular, swollen region in very small truncated – 9 × 15 µm, navicular, – Biconical with short – 3 µm 3 µm 5–7 µm 3 types of sporocysts granules 6–14 μm Spherical, 0.6–1.6 µm 5.8 (±0.2) × 3.5 µm – – – 7 µm 30 µm µm 11 central a central eccentric eccentric vesicular Spherical Eccentric karyosome 2.4–5.7 µm – × 8 µm 12–17 60 µm 10 µm Nucleus Karyosome Paraglycogen 8–12 µm 8–16 µm eccentric 30–90 µm 9–36 µm Voluminous Voluminous around nucleus ones toward the 31 µm vacuoles Mainly eccentric granules near the center of nucleus. nuclear membrane and irregular small spherical chromatic infecting some earthworms in world. free. 46 µm 60 µm 50 µm Round, 500 µm 160 µm 40 µm to (57 ± 19) 48–65 µm 42–83 µm 84–450 µm Trophozoite 225–295 µm 57.7 µm (±6.3µm) Thin layer of host’s Thin layer of host’s Young in amoebocyte Young groups attached to the amoebocytes, in small body wall of host, rarely Apolocystis and Pheretima hawayana Pheretima (Coelom behind clitellum region, occasionally in nephredia) Allolopophora terrestris in ovisac, troph. (Young free in the coleomic cavities of the genital segments) Pheretima californica Pheretima elongata P. (Coelomic cavities front of crop region) Dendrobaenarubida Dendrobaenarubida (Lumbricidae) (Perivisceral coelom of genital segments) , L. Lumbricus terrestris castaneus and L. rubellus (Seminal vesicle) Pheretima posthuma Pheretima (Seminal vesicle) Pheretima Pheretima (Parapheretima) beaufortii (Seminal vesicle) Amynthas robusta (Seminal vesicle) Allolobophora chlorotica (Seminal vesicle) Host (Site of infection) A. michaelseni A. laverensis A. proventus A. megagranulata A. minuta A. minima A. beaufortii A. chotonagpurensis A. pertusa Parasite Table 3. Comparison among some species of Table A New Aseptate Gregarine from Egyptian Earthworms 239

A. herculea in the coelomic spaces of earthworm Libyo- drilus violaceus extending from the genital segments to the posterior segments. He added that all different stages in the life cycle were attached with chloragogen cells (splanchnic epithelial layer) of the intestinal wall Armendariz 2002 in the form of groups, and each group was surrounded by very thin transparent membranes. Segun (1971b) described a new species of gregarine, A. megagranu- lata from the perivisceral coelom of genital segments 262 (±44.3) syncytial wall

148.8 (±41.2) × in Dendrobaenarubida sp. This parasite was different from the present parasite by the large size of its paragly- cogen granules (spherical: 6 to 14 μm), and immobility of their trophozoites. The present parasite is the small- est Egyptian species of acephaline gregarine belonging 5–15 µm 2.7–6.8 × to genus Apolocystis (57.7 ± 6 μm). It also represents the first smaller acephaline gregarine, belonging to genus Apolocystis, infecting the coelomic cavities of oligochaetes around the world. Universally, it seems to – be the third species concerning the size of its tropho- zoites after A. minuta Troisi, 1933; (40 μm to 46 μm) and A. beaufortii Cognetti de Martiis, 1918 (Cognetti de Martiis, 1923); (50 μm). It is evident from the above detailed discussion that the gregarine of P. californica vesicular 17 (±7.7), and P. elongata is a new species of Apolocystis, well characterized by its small size, heterogeneity of the endoplasm, obvious thickness of its gametocyst’s wall and the restricted area of infection in the coelom around the pharyngeal region before the crop (proventriculus). eccentric 70–80 µm 6 µm 7–8 µm 32 × 12 µm 1200 × 400 µm Bosanquet 1894 The authors give it the name, Apolocystis proventus sp. 32.8 (±8.413), 37.4 (±11.5) × 37.4 (±11.5) nov. (Table 3). Taxonomic summary Type material: Apolocystis proventus sp. nov. Type host: Pheretima californica and Pheretima elongata. rarely free. Young troph. Young 128.4 (±51.7) embedded into of the intestine 4000 × 3000 µm

mainly free in the Symbiotype: Host is deposited in the Parasitology 158.2 (±63.578) × coelomic cavity or layer, growing and layer, around most stages mature trophozoites attached them to the visceral peritoneum, attached to epithelium Thin mesothelial layer parietal as well to the the splanchnic epithelial Laboratory, Department of Zoology, Faculty of Sci- ence, Ain Shams University, Abbassia 11566, Cairo, Egypt. Site of infection: Pharyngeal glands and peripha- ryngeal coelom. Type locality: Zoological gardens, Giza governorate (Lattitude and Longitude: 30°01′27.3″N 31°12′49.3″E) Lumbricus terrestris Lumbricus rubellus Octocalsium lacteum (Posterior region of the host worm) Microscolex dubius. Microscolex troph. intercellular (Young epithelium, in oesophageal other stages in coelomic cavity along worm, spores in seminal vesicles) and Maghagha region, Menia governorate (Lattitude and Longitude: 28°39′00″N 30°50′24″E), Egypt. Prevalence: Pheretima californica, 6 worm infect- ed out of 43. (13.9%) P. elongata, 7 worm infected out of 23 (30.4%). A. herculea A. janovy 240 N. Ramadan et al.

Type material: Hapantotype and paratypes are de- Loubatières R. (1955) Contribution à l‛étude des gregarinomorphes posited in the Parasitology Laboratory, Department of Monocystidae parasites des oligochètes du Languedoc Roussil- Zoology, Faculty of Science, Ain Shams University, lon. Ann. Sci. Nat. Zool. 17: 73–201 Marek J. (1967) The gregarines found in the seminal vesicles of Abbassia 11566, Cairo, Egypt. Lumbricus rubellus Hoffmeister and Lumbricus terrestris Etymology: The new species has been named after L. from Wroclaw and the neighbourhood. Zool. Pol. 17: 259– the site of infection (restricted to ceolomic cavity front 273 of the crop (= proventriculus) of the host worm). Philips N. E., Mackinnon D. L. (1946) Observations on a monocys- tid gregarine Apolocystis elongata n.sp. in the seminal vesicles of Eisenia foetida (Sav.). Parasitol. 36: 65–74 REFERENCES Pizl V. (1989a) Monocystid gregarines (Protozoa, Apicomplexa) Armendariz L. C., Gullo B. S. (2002) New species of Apolocystis of some Czechoslovak earthworms. Vestnik Ceskoslovenske (Aseptatorina: Monocystidae) from the coelom of Microscolex Spolecnosti Zoologicke 53: 141–152 dubius (Oligochaeta: Acantodrillidae) in Los Talas, Buenos Ai- Pizl V. (1989b) An addition to the Czechoslovak records of res, Argentina. Acta Protozool. 41: 407–413 monocystid gregarines. Vestnik Ceskoslovenske Spolecnosti Bandyopadhyay P. K., Roychudhuri U. S., Biswas G. (2004) De- Zoologicke 53: 226–232 scriptions of Two New Species of Acephaline Gregarines (Pro- Pizl V. (1990) The occurrence of monocystid gregarines in some tozoa: Apicomplexa: Eugregarinida), Apolocystis chotonagpu- Polish earthworms. Acta Protozool. 29: 353–364 rensis sp. n. and Stomatophora janovyi sp. n. from Earthworms Pradhan D., Dasgupta B. (1983) New acephaline gregarines (Annelida: Oligochaeta) of India. Acta Protozool. 43: 275–279 (Apolocystis) in the hill areas of Darjeeling district. J. Beng. Bhatia B. L., Setna S. (1926) On some more gregarine parasites of Nat. Hist. Soc. (NS) 2: 5–12 Indian earthworms. Arch. Protistenkd. 53: 361–377 Ramadan N. F. (1969) Acephaline gregarines in Egyptian oligo- Bhowmik B., Bandyopadhyay P. K., Mitra A. K. (2012) Apolocystis chaetes. M.Sc., Faculty of Science, Ain Shams University, cognetti sp. nov. (Apicomplexa: Monocystinae) a new aseptate Egypt gregarine species from the earthworm Amynthas hawayanus Rees B. (1963) Studies on monocystid gregarines. Six Apolocystis (Annelida: Oligochaeta) from West Bengal. J. Parasit. Dis. 36: species including four new species A. lavernensis, A. perfida, 203–206 A. rotaria, A. spinosa. Parasitol. 53: 491–500 Boisson C. (1957) Monocystidae parasites d’oligochètes d’Indochine. Ruston J. (1959) Dirhynchocystis minuta n. sp., gregarine from the Ann. Sci. Nat. Zool. 19: 72–90 seminal vesicles of Lumbricus terrestris L., with a note on the Bosanquet W. C. (1894) Notes on a gregarine of the earthworm association of Rhynchocystis porrecta Schmidt. J. Parasitol. Lumbricus hercules. Quart. J. Micr. Sci. 36: 421–433 (Cited 45: 259–262 from Loubatières, 1955) Segun A. O. (1971a) Acephaline gregarines of British earthworms- Cognetti de Martiis L. (1923) Sul genere Monocystis. Monit. Zool. their possible host specificity. Parasitol. 62: 389–396 Ital. Firenze 34: 250–253 Frolov A. D. (1991) The World Fauna of Gregarines, Family Mono- Segun A. O. (1971b) Acephaline gregarines of earth worm additions cystidae. Trudy Zoologicheskogo Instituta Akademii Nauk to the British record. J. Protozool. 18, N2: 313–317 SSSR. No. 229, 125 pp. Segun A. O. (1978) Monocystid gregarine parasites of Nigerian Hesse E. (1909) Contribution a l’étude des monocystidées des oli- earthworms. J. Protozool. 25: 157–162 gochètes. Arch. Zool. Exp. Gen. 43: 27–301 Troisi R. A. (1933) Studies on the acephalines of some oligochaete Levine N. D. (1977) Revision and checklist of the species of the annelids. Trans. Amer. Micr. Soc. 52: 326–352 aseptate gregarines family Monocystidae. Folia Parasitol. 24: 1–24 Levine N. D. (1988) The protozoan phylum Apicomplexa Vol. I and Received on 12th April, 2014; revised on 27th October, 2014; ac- II. CRC Press Inc. Florida U.S.A., 15–42 cepted on 5th November, 2014